slc1a5 mediates glutamine transport required for …...2013/01/10 · human cancer biology slc1a5...

Human Cancer Biology

SLC1A5 Mediates Glutamine Transport Required for LungCancer Cell Growth and Survival

Mohamed Hassanein1, Megan D. Hoeksema1, Masakazu Shiota2, Jun Qian1, Bradford K. Harris1,Heidi Chen3, Jonathan E. Clark5, William E. Alborn5, Rosana Eisenberg4, and Pierre P. Massion1,6,7

AbstractPurpose: We have previously identified solute-linked carrier family A1 member 5 (SLC1A5) as an

overexpressed protein in a shotgun proteomic analysis of stage I non–small cell lung cancer (NSCLC) when

compared with matched controls. We hypothesized that overexpression of SLC1A5 occurs to meet the

metabolic demand for lung cancer cell growth and survival.

Experimental Design: To test our hypothesis, we first analyzed the protein expression of SLC1A5

in archival lung cancer tissues by immunohistochemistry and immunoblotting (N ¼ 98) and in cell lines

(N¼ 36). To examine SLC1A5 involvement in amino acid transportation, we conducted kinetic analysis of

L-glutamine (Gln) uptake in lung cancer cell lines in the presence and absence of a pharmacologic inhibitor

of SLC1A5, gamma-L-Glutamyl-p-Nitroanilide (GPNA). Finally, we examined the effect of Gln deprivation

and uptake inhibition on cell growth, cell-cycle progression, and growth signaling pathways of five lung

cancer cell lines.

Results: Our results show that (i) SLC1A5 protein is expressed in 95% of squamous cell carcinomas

(SCC), 74% of adenocarcinomas (ADC), and 50% of neuroendocrine tumors; (ii) SLC1A5 is located at the

cytoplasmicmembrane and is significantly associatedwith SCChistology andmale gender; (iii) 68%ofGln

is transported in a Naþ-dependent manner, 50% of which is attributed to SLC1A5 activity; and (iv)

pharmacologic and genetic targeting of SLC1A5 decreased cell growth and viability in lung cancer cells, an

effect mediated in part by mTOR signaling.

Conclusions: These results suggest that SLC1A5 plays a key role in Gln transport controlling lung cancer

cells’ metabolism, growth, and survival. Clin Cancer Res; 1–11. �2012 AACR.

IntroductionLung cancer is the leading cause of cancer-related deaths

in the United States (1). Non–small cell lung cancer(NSCLC) accounts for about 80% of all lung cancers.Although advances have been made in the diagnosis andtreatment strategies in the last decade, the prognosis ofpatients with NSCLC remains poor, with a 5-year overallsurvival of 15% to 20% (2). A small subset of tumors hasbeen found to be driven by mutated oncogenes for whichactive, but still noncurative, therapies are available. Yet, the

vast majority of tumors have a complex pathogenesis that isstill poorly understood (3). Thus, newmolecular diagnosticand therapeutic targets are urgently needed to improve thequality of care and survival of patients with lung cancer.Although genomic profiling has given important newinsights into themechanisms of carcinogenesis, therapeutictargets and most biomarkers with clinical use are proteinproducts. Advances in proteomic techniques in recent yearshave allowed for an in-depth analysis of the changes inprotein expression and posttranslational modificationsassociated with lung cancer (4, 5). These studies haveyielded large inventories of proteins that can potentiallybe translated to molecular targets or biomarkers of lungcancer, but few of these candidates have been validated orassociated with functional relevance to the disease process.

Given the urgent need for a reliable and noninvasivediagnostic test for lung cancer, we have previously com-pared shotgun proteomic profiles of fresh-frozen stage INSCLC to matched control lung specimens and identifiedseveral candidates that were significantly overexpressed inNSCLC (6). Solute linked carrier family 1 member A5(SLC1A5) emerged as a top candidate. SLC1A5 acts as ahigh-affinity transporter of L-glutamine (Gln) in rapidlygrowing epithelial and tumor cells in culture (7). Neutralamino acids, including Gln, can be transported by 4 main

Authors' Affiliations: 1Division of Allergy, Pulmonary and Critical CareMedicine, Department of Medicine; Departments of 2Molecular Physiologyand Biophysics, 3Biostatistics, and 4Pathology; 5Jim Ayers Institute ofPrecancer Detection and Diagnosis, Vanderbilt University School of Med-icine; 6Department of Cancer Biology, Vanderbilt-Ingram Cancer Center;and 7Veterans Affairs, Tennessee Valley Healthcare System, NashvilleCampus, Nashville, Tennessee

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/)

Corresponding Author: Pierre P. Massion, MD Vanderbilt Ingram CancerCenter, PRB 640, 2220 Pierce Avenue, Nashville, TN 37232. Phone: 615-936-2256; Fax: 615-936-1790; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-2334

�2012 American Association for Cancer Research.

ClinicalCancer

Research

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families of amino acid transporter systems including sodi-um-dependent systems A, ASC, N, and sodium-indepen-dent system L (8–11). These transporters are classified onthe basis of tissue distribution and affinities for differentamino acids. System ASC is the most commonly expressedamino acid transporter in human tumor-derived cells, sug-gesting it might play a major role in cellular transformationand mediation of Gln-dependent growth (10, 12, 13).Because SLC1A5 belongs to the Naþ-dependent ASC familyof amino acid transporters, we examined whether gluta-mine is transported in lung cancer cells in aNaþ -dependentmanner. In lung cancer, the dependency on Gln for growth,survival, and cell-cycle progression iswell documented (14–16). More recently, Gln conversion into the tricarboxylicacid cycle intermediate a-ketoglutarate through glutamin-ase was shown to be essential for Kras-induced anchorage-independent growth in A549 cells (17). Other studiesshowed that reductive carboxylation of glutamine is keyfor the metabolic reprogramming that enables cancer cellsto survive and proliferate under hypoxia in several cancercellular models, including lung cancer (18–20). Collective-ly, these studies provide strong evidence for a major role ofGln metabolism in supporting lung cancer cell growth andsurvival. However, the identity of the transporter(s) thatcapture Gln in lung cancer cells and how Gln transport islinked to cell growth and survival is still largely unknown.Therefore, the 3 main objectives of this study were first, toevaluate the expression pattern of SLC1A5 protein in dif-ferent lung cancer pathologic subtypes, second, to assess itsrelevance to the clinical outcomes in lung cancer, andfinally, to investigate the functional contribution of SLC1A5to glutamine uptake and its role in supporting the cellgrowth and viability of lung cancer cells.

Materials and MethodsPatient selection and tissue microarray

Tissuemicroarrays (TMA) of 98 lung cancer tumor tissueswere prepared from paraffin-embedded formalin-fixed(FFPE) blocks. The TMA consisted of 46 adenocarcinomas,37 squamous cell carcinomas, 4 bronchioalveolar carcino-mas, 4 large cell carcinomas, 2 NSCLC, 5 carcinoids, and 1each of adenosquamous, sarcoma, and small cell lung

cancer (SCLC). These arrays were constructed according toprotocols previously described (21). Archived tissueblocks from consecutive anatomic resections between1989 to 2002 were retrieved from the files of the VanderbiltUniversity Medical Center and the Nashville VeteransAdministration Medical Center pathology departments(Nashville, TN). Demographic and clinical characteristicsof the 98patients represented inbothTMAsare summarizedin Table 1, for details in immunohistochemical (IHC)analysis, refer to Supplementary Materials.

Cell cultureHuman lung cancer cell lines A549 (ADC), H1819

(ADC), HCC15 (SCC), H520 (SCC), andH727 (Carcinoid;American Type Culture Collection) were maintained inRPMI-1640medium (Gibco by Life Technologies) contain-ing 10%heat-inactivated FBS (Gibco by Life Technologies),at 37�C, 100% humidity, and 5%CO2. Cells were passagedevery 2 to 3 days to maintain exponential growth.

Glutamine uptake assaysMeasurement of Gln uptake in monolayer cultured

NSCLC cells was carried out via the cluster-tray methodoriginally described byGazzola and colleagues (22). Briefly,cellswere plated at 105 cells perwell in 24-well culture plates(Costar) and allowed to adhere overnight. Before transport

Translational RelevanceWeherein report for thefirst time the clinical relevance

of SLC1A5 as a diagnostic biomarker and its biologicfunction in lung cancer. Our results show that SLC1A5mediates glutamine transport that is required for lungcancer cell growth and survival. These findings identifySLC1A5 as a primary transporter of glutamine in lungcancer cells, helping to explain their growth dependencyon glutamine. The enhanced cell surface expression ofSLC1A5, its activity as an amino acid transporter, and itsvital role in supporting lung cancer growthmake it a newpotential diagnostic and therapeutic target.

Table 1. Characteristics of the patients in theTMAs cohort according to the cancer status andSLC1A5 expression

Total SLC1A5 SLC1A5N ¼ 98 (�) (þ)

Patients demographics n (%) n (%) n (%)

Age, Mean (SD) 56.4 � 11.5RaceAfrican American 7 (7) 0 7 (100)Caucasian 91 (93) 17 (17) 81 (83)

GenderMen 56 (57) 6 (11) 50 (98)Women 42 (43) 13 (31) 29 (69)

Smoking statusEx. never-smoker 71 (72) 13 (18) 56 (82)Current-smoker 27 (28) 3 (11) 24 (88)

Pack-years, mean (SD) 55.78 � 33HistologyADC 46 (47) 12 (26) 34 (74%)SCC 37 (38) 2 (5) 35 (95%)Other NSCLC 9 (9) 0 9 (100%)Neuroendocrinetumors

6 (6) 3 (50) 3 (50%)

Abbreviations: Neuroendocrine tumors, SCLC þ large cellNeuroendocrineþ Atypical carcinoid; Other NSCLC, LCCþadenosquamous carcinoma þ NSCLC; PKY, pack year(pack per day � number of years smoked).

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assays, the cells were rinsed twice with warm Naþ-freeKrebs-Ringer Phosphate Buffer (cholKFtP) inwhich cholinechloride and choline phosphate iso-osmotically replacedthe corresponding Naþ salts, to remove extracellular Naþ

and amino acids. The radiotracer used was L-[3,4Gln-3H]glutamine (Amersham) at 500,000 dpm/mmol of the spe-cific activity (5 mCi/mL). For kinetic studies, the amount ofunlabeled glutamine in the transport buffer varied from400mmol/L to 6.4 mmol/L. Transport values were obtainedeither in the absence of extracellular Naþ (diffusion andNa-independent uptake) using cholKRP or in the presenceofNaþ (total uptake), usingNaKRP buffer to determine theNa-dependent rates, reported in units of picomoles permilligram protein per min. All transport measurementswere carried out at 37�C and were terminated after 3minutes by adding ice-cold PBS saline (PBS) followed by3 rapidwasheswith an ice-cold PBS. Intracellular glutaminewas extracted with 0.2 mL per well of 0.2% SDS in 0.2 NNaOH; after 1 hour at room temperature, 0.1 mL of thelysate was neutralized with 2N HCl and subjected to liquidscintillation spectrophotometry. The remaining lysate wasused for the determination of cellular protein by the PierceBCA Protein assay. Rates of glutamine transport were cal-culated from the counts per minute (cpm) per sample, andthe specific activity of the uptake mix (in cpm/nmol). Theseresults were then normalized to cellular protein contentusing Microsoft Excel. For pharmacologic targeting by g-L-Glutamyl-p-Nitroanilide (GPNA), Gln uptake assays wereconducted in the presence of 0, 100, and 300 mmol/L ofGPNA in NaKRP buffer. The transport velocities were cal-culated from radioactive counts and protein concentrationand subsequently expressed as pmol of Gln transported permilligram of protein perminute. Each data point representsthe average � SEM of at least 3 separate determinations.

Glutamine-dependent proliferation assaysTo test the growth dependency on Gln, cells were plated

in 12-well tissue culture plates at a density of 2 � 104 cellsper well. The following day, the cells were rinsed once withserum-freeGln–freemediumand replacedwith RPMI-1640(Gibco by Life Technologies) with the following variations:supplementionwithEGF (25ng/mL) and1� growth factorscocktail (Invitrogen) that includes insulin, selenium, andtransferein (Sup), Supþ2mmol/LGln (þGlnþSup), RPMI-1640þ Gln, but no supplements (þGln�Sup), no Gln butwith supplements (�GlnþSup) or with neither Gln norsupplements (�Gln�Sup). Cultures were left to grow for 3days with media changes every 48 hours. To test whetherSLC1A5 activity mediates the Gln depletion effect, A549and H520 were cultured in media containing (þGlnþSup)þ 1 mmol/L GPNA, and left to grow for 2 days. To furthervalidate the antigrowth effects of SLC1A5 blockade byGPNA,A549 andH1819 cellswere cultured in the optimumgrowth media (þGlnþSup) in the presence of increasingdoses of GPNA (0, 1, 10 100, 1,000 mmol/L) for 2 days. Cellgrowthwasmonitored bymeasuring theOD490 by the Cell-Titer 96-Aqueous colorimetric assay (Promega) at day 0and after 48 hours of culture. Relative growth rates were

expressed as%growth fromday 0 andwere calculated usingthe following equation; (Ti-Tz)/(C-Tz) � 100, where Ti isthe cell number after 48 hours (i¼ inhibition), Tz is the cellnumber at time 0 and C is the cell number of the controlcells that were cultured in optimum growth conditions(þGln þSup; ref. 23).

Transfection of siRNAAmixture of four 21-nucleotide siRNAs that target human

SLC1A5 was synthesized (Thermo Scientific) and was pro-vided as single reagent. The 4 siRNA sequences targetinghuman SLC1A5 in the siRNA pool were;

1) UGAUACAAGUGAAGAGUGA,2) GCAAGGAGGUGCUCGAUUC,3) GGUCGACCAUAUCUCCUUG,4) GCCUUUCGCUCAUACUCUA.

A nontargeting control siRNA of scrambled nucleotidesequence used as a negative control for nonspecific binding(siRNACont) was purchased from the same company. All-Stars Hs Cell Death Control siRNA was used as a positivecontrol to verify both the transfection efficiency and as apositive control for cell viability (Qiagen). A549 and H520cells were transfected usingDharmaFECT1- reagent (0.2 mL/well) at 1.0 and 2.5 nmol/L siRNAs for 72 hours beforeassessing cell growth, viability, and cell-cycle progression asdescribed in Supplementary Materials.

Statistical analysisThe association between SLC1A5 expression and clinical

variables was analyzed using the Kruskal–Wallis or Wil-coxon rank-sum tests. Kinetic data was fitted to Michaelis–Menten kinetics with data points equal to mean� SEM of nexperiments minus nonspecific binding. Statistical analysisfor the glutamine uptake kinetics and cell growth assays wasconducted with GraphPad Prism (GraphPad Software).Data comparing 2 experimental conditions were statistical-ly analyzed by 2-tailed Student t test and only results with P< 0.05 were considered to be statistically significant. Allexperimental data are presented as the mean � SEM of nindependent measurements (as indicated in each figurelegends). All treatments within each experiment were con-ducted in triplicate wells and repeated on 3 independentdays. To assess the extent of the dose response treatment ofGPNA, ordinary regression analysis was used to evaluate thelinear trend between log and log (percent of growthinhibition).

ResultsSLC1A5 is overexpressed at the cytoplasmic membraneand is associated with squamous lung cancer histologyand male gender

Our shotgun proteomic analysis showed that SLC1A5protein is overexpressed in tissue extracts of both stage Iadenocarcinoma (ADC; N ¼ 20) and squamous cell carci-noma (SCC; N ¼ 20) of the lung compared with controllung samples (N ¼ 20) from fresh-frozen tissue and from

Investigating the Biological Function of SLC1A5 in Lung Cancer

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formalin-fixed paraffin-embedded FFPE tissues (N ¼ 5 ofeach tissue type; Supplementary Fig. S1A and S1B). Weconfirmed these results in a separate set of tissue lysatescollected from 3 normal lung tissues and 3 NSCLC tumors(Supplementary Fig. S1C). To evaluate the pattern of expres-sion for SLC1A5 protein in lung tumors, we conductedimmunohistochemical (IHC) analysis using 2 TMAs ofarchival primary lung cancer tissues collected from patientsdiagnosed with different histologic subtypes of lung cancerusing a validated human anti-SLC1A5 IgG (http://www.proteinatlas.org/). SLC1A5 signals were seen in 35 of 38(95%) of SCC and of 34 of 46 (74%) ADC subtypes. OtherNSCLCs, including large cell carcinoma (LCC), adenosqua-mous, andNSCLC, not otherwise specified were all positive(9/9; 100%). Neuroendocrine tumors, which includedatypical and typical carcinoid and SCLC, were also repre-sented in our TMA but only 50% (3/6) stained positive for

SLC1A5 (Table 1). The pattern of SLC1A5 staining in bothadenocarcinomas and squamous cell carcinomas was alongthe cytoplasmic membrane, with less intense cytoplasmicstaining. Of the normal cellular components, we found 0 to1þ cytoplasmic membrane staining of ciliated respiratoryepithelial cells, 2þ to 3þ cytoplasmicmembrane staining ofbasal bronchial/bronchiolar cells, 2þ cytoplasmic mem-brane staining of bronchial submucosal glandular cells, 1þto 2þ cytoplasmic membrane staining of reactive type 2pneumocytes (but no staining in type 1 pneumocytes), 2þcytoplasmic staining of alveolar macrophages, and 1þ to2þ cytoplasmic and cytoplasmic membrane staining ofplasma cells. There was no staining of stromal fibroblasts,smooth muscle, cartilage, endothelial cells, or lymphocytes(Fig. 1A). The occurrence of SLC1A5 expression was signif-icantly higher in SCC than in ADC (P < 0.001; Fig. 1B). Themost significant correlation between SLC1A5 and clinical

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Figure 1. SLC1A5 is differentially expressed in NSCLC tumors and located at the level of the cytoplasmicmembrane. A, representative images of IHC stainingfor SLC1A5 protein in a TMA constructed from archival lung tissue sections collected from 98 patients with lung cancer representing the main lungcancer histologic subtypes. Left, 3 representative photomicrographs at � 100 magnification of sections of normal lung tissue from patients with lungcancer, which stained negative for SLC1A5. Right, representative photomicrographs at� 100magnification of lung cancer sections from patients with stageIIA ADC and IIB SCC. A zoomed in � 200 magnification of a small area of the same sections in the top right corner shows membranous stainingpattern of cancer cells. A section of ADC that did not stain for SLC1A5 is also represented here. B, a box plot of SLCA15protein IHC index (intensity�%tumorcells) shows significantly higher index of staining (P < 0.001) of tumors in males (N ¼ 56) than in females (N ¼ 42). C, a box plot of SLCA15 protein IHC indexshows significantly increased IHC staining index in SCC compared with ADC (P < 0.001). A detailed summary of our IHC analysis is provided in Table 1.

Hassanein et al.


variables was observed with gender, being higher in malesthan females (P < 0.001; Fig. 1C) and with histologicsubtype, being higher in SCC than ADC (P < 0.001; Fig.1C and Table 1). No significant correlation was observedwith SLC1A5 expression and the overall survival, pack yearhistory of smoking or age.The immunohistochemistry results from lung cancer cell

lines showed that all but one adenocarcinoma cell line(H2009; Supplementary Table S1, not shown in immuno-histochemistry) and one SCLC cell line (H345) stainedpositive for SLC1A5 (Supplementary Table S1 and Supple-mentary Fig. S2A). Similar to lung primary tumors, wefound that the subcellular location of SLC1A5was predom-inantly membranous (Supplementary Fig. S2A) in all lungcancer cell lines. Western blot analysis using a rabbit poly-clonal anti-SLC1A5 antibody (24, 25) confirmed that theexpression of this protein is higher inmalignant lung cancercell lines, A549 (ADC), HCC15 (SCC), H520 (SCC), andH460 (LCC; Supplementary Fig. S2B and S2C). The spec-ificity of this antibody and the confirmation of the SLC1A5expression in A549 andH1819 cell lines were verified using3 different antibodies (Supplementary Fig. S3A). One ADCcell line, (H1819), was negative for SLC1A5. H727, acarcinoid cell line, had low levels of the protein. TheWestern blot results from lung cancer cell lines are consis-

tent with the pattern of expression of SLC1A5 from shotgunproteomics (Supplementary Fig. S1A–S1C) and fromimmunohistochemistry of tumor TMA (Fig. 1A). Theseresults show strong cell membrane immunostaining forSLC1A5 in most lung cancers, more so in SCC and in men.

Glutamine uptake in lung cancer cells is mediated inpart by SLC1A5

We tested the Naþ dependency of the cellular uptake ofGln in A549 cells using L-[G-3H] in Krebs–Ringer solution(Fig. 2A and B; ref. 26). Our results depicted in Fig. 2A andSupplementary Table S2 showed that 68% of cellular Glnuptake occurs in a Naþ-dependent manner. To test thecontribution of SLC1A5 in Gln uptake by lung cancer cells,we conductedGln uptake assays in A549 cells in the absenceor in the presence of the glutamine analogue, GPNA, acompetitive inhibitor of Gln uptake that binds specially toSLC1A5 (27, 28).Glnuptake kinetics inA549 cells showedadose-dependent inhibition of Gln uptake of 15% (P < 0.05)and 32% (P < 0.005) by incubating at 100 and 300 mmol/Lof GPNA for 3 minutes, respectively (Fig. 2B). No furtherinhibition ofGlnuptakewasobservedwhen concentrationsof GPNA were increased up to 900 mmol/L under the sameconditions (data not shown). These results suggest that themajority of the cellular Gln uptake in A549 occurs in a Naþ

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Figure 2. Gln uptake isNaþ-dependent andmediatedpartially bySLC1A5. Toexamine if intracellular transportation ofGln, humanADCA549cellswere seededat the indicated cell densities into 24-well culture plates (0.5 mL/well). A, the Naþ-dependent uptake of 1.6 mmol/L of Gln was monitored for 3 minutesat 37�C. Each point represents the average � SEM for quadruplicate determinations. This figure also illustrates the Michaelis–Menten kinetics ofglutamine rates in cholKRP (�Naþ) or NaKRP (þNaþ; A, top right), and (þNaþ) > (�Naþ).��,P < 0.005 (n¼ 3). B,Vmax values of Gln uptake kinetics of A549 cellsin the presence of 0, 100, and 300 mmol/L GPNA and the top right panel shows the Michaelis–Menten kinetics of glutamine rates. C, dose–responseinhibition of%growth of A549 after incubation in full-growthmedia (þGlnþsupp) containing increasing doses of GPNA. D, the intracellular ROS levels in A549cells were measured by measuring the fluorescence signal of H2 DCFDA (Ex488nm/Emiss525nm) using microtiter plate reader after 24 hours of incubation inincreasing doses of GPNA. These data are representative of at least 3 independent observations and results are average � SEM.



-dependent manner and that approximately 50% of it ismediated by SLC1A5. To test the effect of pharmacologicblockade of SLC1A5-mediated Gln uptake in lung cancercell growth, we incubated A549 cells in the presence ofincreasing concentrations of GPNA for 48 hours. A dose–response growth inhibition was observed (Fig. 2C) com-pared with growth of cells without Gln and supplement forthe same duration. These results present the first evidencethat targeting SLC1A5 activity in lung cancer cells candirectly affect cell growth.

SLC1A5 expression regulates growth dependency onglutamine in NSCLC cells

The role of SLC1A5 expression in regulating glutaminemetabolism-dependent lung cancer cell growth has notbeen investigated. We therefore cultured 5 lung cancer celllines that varied in their level of protein expression ofSLC1A5 (Fig. 3A and Supplementary Fig. S3), and culturedthem in media that varied in Gln and growth factorsconcentrations for 72 hours as described in the Methods

section. Under Gln-deprived condition, cell growth wassignificantly decreased in cell lines that had high levelsof SLC1A5 expression (A549, HCC15, and H520), butno significant effect was observed in cell lines thathad low (H727) or undetectable levels of SLC1A5(H1819; Fig. 3B). Interestingly, H1819 (SLC1A5 null)grew much slower even under optimum growth condi-tions (þGlnþSup) compared with A549, H520, andHCC15, all of which overexpress SLC1A5. Pharmacologictreatment of A549 cells (overexpresses SLC1A5) withincreasing doses of 6-diazo- -oxo-L- norleucine (DON),a glutamine antagonist (29) for 4 days resulted in signif-icant growth inhibition in these cells, whereas H1819(SLC1A5 null) was unaffected (Fig. 3E). These resultssuggest that SLC1A5 expression regulates at least in partlung cancer cell growth dependency on glutamine. Toexamine the specific contribution of SLC1A5 in lungcancer cell dependency on glutamine, we treated A549and H520 cells (overexpress SLC1A5) with 1 mmol/LGPNA for 48 hours. Our results depicted in Fig. 3C and

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Figure 3. Glutamine is required for growth of lung cancer cells in vitro. To test whether SLC1A5 expression is correlated with Gln-dependent growth of lungcancer cells in vitro, 5 lung cancer cell lines that vary in their expression level of SLC1A5 protein (A) were grown in culture media that are supplemented withEGF, insulin, selenium and transferein, and 2 mmol/L of Gln (þGlnþSup), or Gln but no supplements (þGln�Sup), or no Gln but with supplements(�GlnþSupp) or not supplemented with neither Gln not supplements. B, the fold change in cell growth was analyzed at day 3 by Cell-Titer 96-AqueousColorimetric Assay asa change of optical density (OD) at 490 nmsignal fromday 0. Sensitivity of growth inhibition toGln deprivationwas significant in cell linesthat overexpress SLC1A5, A549, H520, and HCC15, but not in cell lines that have low or undetectable level of the protein H1819 and H727. C, the effectof Gln deprivation on cell viability in SLC1A5-expressing cell lines A549 and H520 were measured by Trypan blue Exclusion Dye method after48 hours of culturing in media that either supplemented or deprived of Gln. D, the antigrowth effect of Gln deprivation was measured by calculating the netincrease or decrease of cellular growth after 48 hours of culturing under media that was either supplemented or deprived of Gln or fully supplementedmedia þ 1 mmol/L of GPNA using the following equation: (Ti-Tz)/(C-Tz) � 100. Where Ti is the cell number after 48 hours of treatment of growth stimuli orinhibitors (i¼ inhibition), Tz is the cell number at time zero, and C is the cell number of the control cells that were cultured in optimum growth conditions (þGlnþSup). E, dose–response decline in cell growth after treatment of A549 andH1819with increasing concentrations of DON for 48 hours. Statistical significancewas assessed by Student t test and was denoted as �, P � 0.05; ��, P � 0.005 from 3 independent assays.

Hassanein et al.


D show a significant decrease in cell growth in both cellA549 and H520 cell lines, whereas viability was lessaffected.

Blockade of SLC1A5-related Gln uptake in lung cancercells increases release of intracellular ROSOur shotgun proteomic data from fresh-frozen tissue of

stage I lung cancer showed an increased expression of bothalanyl amino peptidase (ANPEP) and glutathione synthe-tase (GSS), 2 key enzymes in the glutathione synthesispathway (data not shown). Because glutamine acts as aprecursor in the biosynthesis of glutathione (GSH; ref. 30)and because GSH functions as a scavenger for intracellularexcess of reactive oxygen species (ROS; refs. 31, 32), wetested whether inhibiting SLC1A5-mediated Gln uptake inA549 cells would increase the intracellular level of ROS.When treated with increasing doses of GPNA for 24 hours,A549 cells exhibited rising levels of intracellular ROS asmeasured by the relative fluorescence signal of the oxidizedformofH2DCFDA (Fig. 2D). To testwhether the antigrowtheffect and the increase of intracellular ROS release weremediated by SLC1A5-Gln activity, wemeasuredGln uptake,cell growth, and intracellular ROS release in A549 cellswhich express high levels of SLC1A5 compared withH1819 (Supplementary Fig. S2B). The Gln uptake ofH1819 was significantly lower (by 40%; P < 0.005) thanthat ofA549andwas comparablewith the sameuptake levelof A549 cells when treated with SLC1A5 inhibitor GPNA at300mmol/L (P<0.05).GPNAdidnot affect ofGln uptake inH1819 compared with A549 cells, which are inhibited by33% at 300 mmol/L. Pharmacologic blockade of SLC1A5with increasing doses of GPNA for 48 hours caused a dose-dependent increase in intracellular ROS release in A549 butnot in H1819 (Supplementary Fig. S3C). Similarly, a dose-dependent decrease of cell growth was observed in A549(overexpressing SLC1A5) but not in H1819 (SLC1A5 null)

when cells were cultured in media containing increasingconcentrations of SLC1A5 inhibitor GPNA (SupplementaryFig. S3D). Collectively, these results suggest that Gln uptakeis mediated in part by SLC1A5 transport activity in lungcancer cells and that blockade of this activity decreases cellgrowth and increases release of intracellular ROS.

Targeting SLC1A5 causes G1 arrest by inhibiting mTORsignaling

To evaluate the effects of glutamine depletion and glu-tamine-dependent uptake by SLC1A5 on cell-cycle progres-sion, A549 cells were cultured for 48 hours with eithersupplemented growth media (þGlnþSup), growth supple-ment-depleted media (þGln�Sup), glutamine-depletedmedium (�GlnþSup), media that lacked both glutamineand growth supplements (�Gln�Sup), or in (þGlnþSup)media þ 5 mmol/L GPNA. Glutamine depletion only(�Gln þSup) resulted in complete inhibition of growth asillustrated by the increased percentage of cells arrested at G1

phase and decreased percentage of cells at S and G2–Mphases but cell viability was unaffected (Fig. 4A and B). Asimilar effect was observed in cells treated with GPNA. Totest whether the antiproliferative effect of Gln depletion canbe attributed to SLC1A5 activity, we targeted SLC1A5 genet-ically for downregulation by using a specific siRNA. Asignificant knockdown of SLC1A5 protein was confirmedby Western blotting after 72 hours incubation with 2different concentrations of anti-SLC1A5 siRNA (Fig. 5A).Similar to Gln depletion and GPNA treatment, a significantreduction in viability and growth were observed whenSLC1A5 was downregulated by siRNA (Fig. 5B and C). Thedownregulation of SLC1A5 by siRNA also resulted in cell-cycle arrest at G1.

Although the literature suggests that increased cell surfaceexpression of SLC1A5 can be explained by cancer depen-dency on glutamine metabolism to support its high

Figure 4. Effect of Gln depletion andSLC1A5 inhibition on cell-cycleprogression. A, representative brightfield (� 20 magnification) images ofA549 cells after 48 hours culturing invarious growth conditions asdescribed below. B, DNA histogramsof cell-cycle phase distribution of 104

cells after 48 hours of culturing A549cells in growth media supplementedwith EGF, insulin, selenium andtransferein, and 2 mmol/L of Gln(þGlnþSup), or Gln but nosupplements (þGln�Sup), or no Glnbut with supplements (�GlnþSupp)or not supplemented with neitherGln nor supplements (�Gln�Supp),or with (þGlnþSup) and that contain5 mmol/L of GPNA.

+Gln+Sup

+Gln+Sup

G1 G1 G1 G1 G1

Dip G1Dip G2Dip S

G2 G2 G2 G2 G2

+Gln+Sup+GPNA

S S S S S

Channels (FL2-A)

Co

un

t

+Gln–Sup

A

B

–Gln+Sup

–Gln–Sup

+Gln–Sup –Gln–Sup +Gln+Sup+GPNA–Gln+Sup



demand for energy and macromolecular biosynthesis(33–35), more recent studies revealed that SLC1A5 medi-ation of glutamine transport is independent from gluta-mine metabolism and necessary for activating criticalsurvival and cellular growth signaling cascades includingmTOR, and ERK pathways in some cancer types(24, 28, 36). To test whether Gln uptake into lung cancercells induces mTOR signaling, we first starved H520 cellsof Gln and growth factors for 24 hours. The next day, cellswere supplemented the medium with glutamine (þGln-Sup) or glutamine-free (�GlnþSup) but with growthfactors (EGF and Insulin) or full growth media þ 5mmol/L GPNA for 1 hour. Cells were then harvested,lysed, and the status of mTOR pathway activation wasanalyzed by Western blotting. As shown in Fig. 6B, thelevel of phosphorylated mTOR (p-mTOR) was reducedcompared with total mTOR protein by Gln depletion orGPNA treatment. The reduction of the level of p-mTORresulted in a decrease in the activation of its downstreamtargets, p85 S6K and p70 S6K compared with their totalprotein levels. In contrast with mTOR activation, we didnot detect a significant change in either Akt or ERKsignaling cascades as indicated by the level of phsophory-lated pAKT and p-ERK levels (Fig. 6B). Contrary to H520,signaling through mTOR were unaffected by either Glndeprivation of GPNA treatment in H1819 cells (SLC1A5null), whereas deprivation of growth supplements atten-uate both mTOR and ERK activation in this cell line (Fig.6A). These results are consistent with previous studies thatshowed the ability of extracellular glutamine to activategrowth signaling cascades such as mTOR signaling andsuggest that Gln plays a role as a growth signaling stim-ulus (24, 28). Whether this mechanism is universal in allSLC1A5-expressing lung cancer cells is unknown.

DiscussionWe report for the first time the clinical relevance as

candidate diagnostic biomarker and the biologic functionof SLC1A5 in lung cancer. Our results show that SLC1A5mediates glutamine transport required for lung cancer cellgrowth and survival. Specifically, we report that (i) SLC1A5is overexpressed at the cytoplasmic membrane and is asso-ciated with squamous lung cancer histology and malegender, (ii) glutamine uptake in lung cancer cells is medi-ated in part by SLC1A5, (iii) SLC1A5 expression regulatesgrowth dependency on glutamine in NSCLC cells, (iv)blockade of SLC1A5-related Gln uptake in lung cancer cellsincreases release of intracellular ROS, and (v) targetingSLC1A5 causes G1 arrest by inhibiting mTOR signaling.

SLC1A5expression is significantly associatedwithbutnotrestricted to SCC histology (Fig 1 and Supplementary Fig.S1) and is significantly correlated with higher tumor expres-sion levels in men (Fig. 1B and Table 1). The biologicreasons for this gender correlation are not fully understood.One potential explanation is that sex hormones may reg-ulate SLC1A5 and other amino acids transporters mayexplain these findings and deserve further investigation.The expression pattern of amino acid transporters in dif-ferent solid tumors including liver (10, 12), breast (12) andcolon cancer (37) is emerging in the literature. In lungcancer, a recent histologic study in a cohort of 160 NSCLCsfound that Lat1, a sodium-independent amino acid trans-porter was expressed in 79.6% (43/54) of nonadenocarci-noma and in 15.1% (16/106) of adenocarcinomas (38).The same study also showed that the expression of Lat1 issignificantly correlated with markers of glycolysis, angio-genesis, phosphoinositide-3 kinase (PI3K)/Akt, EGF recep-tor (EGFR), and mTOR pathways. Furthermore, the samegroup foundLat1 tobe correlatedwith chemoresistance and

kDa

% V

iab

ility

SLC1A5

siRNA-cont

siRNA-cont

G1SG2

%66%16%18

G1SG2

G1SG2

%87%7%6

%84%10

%6

siRNA-1 nmol/L siRNA-2.5 nmol/LsiRNA-

contsiRNA

SLC1A5

1nmol/L

2.5nmol/L

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% G

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th

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T-con

tsiR

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t1 n

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ont

siRNA-c

ont

1 nm

ol/L

2.5 n

mol

/L

100

75

50

45

2.5 n

mol

/L Figure 5. Genetic targeting ofSLC1A5 affects cell growth andviability. A, Western blot analysisverification of the downregulationof SLC1A5 protein in A549 andH520 cells transfected withscramble siRNA at 25 nmol/L oranti-SLC1A5 with indicatedconcentrations. After siRNA, 72hours of transfection, cell viability(B), and cell growth compared withday 0 (C) were analyzed by directcell count using Trypan Blue dyeexclusion method. D, cell-cycleanalysis was conducted in A549cells after 72 hours of transfectionwith the indicated siRNAconcentrations. The data areshown as mean � SD andrepresent 3 independentexperiments.

Hassanein et al.


poor prognosis in NSCLC (39). Short of functional data,this report provides preliminary evidence for the potentialimportance of amino acids transporters in lung cancerprogression. Future functional studies should be designedto determine the relative contribution of Lat1 to glutamineuptake in lung cancer cells.SLC1A5 has known functions in normal and cancer cells:

as a retroviral receptor during placental development andcancer–endothelial cell fusion in breast cancer (25, 40), andas a neutral amino acid transporter with high affinity forglutamine (11, 41). Earlier studies in lung cancer models invitro and in vivo showed that glutamine is essential for bothgrowth and viability of the cells (14–16). Therefore, wefocused our efforts on investigating the contribution ofSLC1A5 to glutamine transport in lung cancer cell lines.We hypothesized that the enhanced expression of SLC1A5in lung tumors and cell lines could be an adaptationmechanism that enables lung cancer cells to efficientlycapture the overly abundant Gln from the extracellularmilieu. Our results indicate that most of the Gln uptakeby A549 cells occurs in a Naþ-dependent fashion and thathalf of that is mediated by SLC1A5. Pharmacologic andgenetic targeting of SLC1A5 significantly attenuates cellgrowth by forcing the cells to arrest at G1 phase. Because

SLC1A5 can transport aliphatic neutral amino acids includ-ing glutamine (10, 41), we anticipate that a part of thegrowth inhibitory effect of its blockade can be attributed toreducing the uptake of other neutral amino acids. None-theless, because SLC1A5 has high affinity to Gln, which isthe most abundant amino acid in circulation, and becauseSLC1A5 contributes to 50% of the Naþ-dependent Glntransport (Fig. 2 and Supplementary Table S2), SLC1A5activity is most likely responsible for the phenotypic effectsobserved. These data also suggest that otherNaþ-dependenttransporters such as SN1 (SLC38A3) and/or SN2(SLC38A5) may contribute to Gln uptake in cancer cells.Future studies are needed to evaluate the relative contribu-tion of other amino acids transporters in lung cancer.

The antigrowth effect of siRNA downregulation ofSLC1A5 in lung cancer cells shown in our results is consis-tent with previous studies that used antisense methods todownregulate SLC1A5 in liver cancer cell lines (24). Thedirect impact of SLC1A5 downregulation on cell-cycle pro-gression in lung cancer provides the first strong evidencethat SLC1A5 is a link between Gln availability and celldivision. In addition and consistent with the emergingprosurvival role of L-glutamine in cancer progression (18,19), we found that the protein level of GSS, a rate-limiting

57

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p-ERK

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ββ-Actin

ERK

289

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~

42~

42

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45~

Figure 6. SLC1A5-mediatedGln uptake activatesmTORsignaling.mTOR, AKT, and ERK signaling activationwas evaluated usingWestern blot analysis of thephosphorylation of p-mTOR signaling in H520 (SCC) and H1819 (ADC) cell lines in response to Gln deprivation or SLC1A5 blockade. Cells were deprived ofgrowth factors and Gln (starved) for 24 hours and were treated with RPMI-1640 media that were supplemented EGF, insulin, selenium and transferein,and 2mmol/L of Gln (þGlnþSup), Gln but no supplements (þGln�Sup), or no Gln but with supplements (�GlnþSupp), or not supplemented with neither Glnnot supplements, or the full growth media þ 5 mmol/L of GPNA (þGlnþSup) for 60 minutes. A, Gln deprivation or GPNA treatment has no effect onmTORor ERKsignaling inH1819 cells (SLC1A5null). B, inH520 (SLC1A5overexpressed) the activationofmTORsignaling cascadewas attenuated asevidentby the levels of phosphorylatedmTORand the phosphorylation of its p70S6K and p85S6K p. Gln deprivation or GPNA treatment has no effect in either AKT orERK signaling in H520 cells.



enzyme which catalyzes the conversion of g-L-glutamyl-L-cysteine to glutathione (42), was higher in A549 thanH1819. Interestingly, the expression pattern of GSSmirrorsthat of SLC1A5 in these 2 cell lines. This is consistent withour shotgun proteomic data that showed GSS to be over-expressed in both SCC and ADC stage I NSCLC tissuescompared with control counter parts (data not shown). Totest the hypothesis that Gln transported by SLC1A5 con-tributes to GSH synthesis, we inhibited the transporteractivity with GPNA. Blockade of SLC1A5 resulted in anincrease of intracellular ROS release in A549 (SLC1A5positive), but not inH1819 (SLC1A5negative)with increas-ing doses of GPNA (Supplementary Fig. S3C). These resultssuggest that SLC1A5 expressionmay be one component of awider metabolic reprogramming scheme that is adopted bylung cancer cells to combat oxidative stress in theirmicroenvironment.

Recent studies revealed a new role of SLC1A5 activityindependent from glutaminemetabolism and necessary foractivating critical survival and cellular growth signalingcascades includingmTORandERKpathways in somecancertypes (24, 28). This role for Gln and its transporter in cancercells goes beyond traditional metabolic functions of aminoacids. Consistent with these reports, our results (Fig. 6B)suggest that glutamine uptake via SLC1A5 can activatemTOR signaling independent from growth factors inH520 SCC cells. Altogether, the results of this study suggestthat growth dependency on glutamine is linked to SLC1A5expression and activity and that inhibition of its Gln uptakeactivity has cytostatic effects in lung cancer cells in vitro.

In conclusion, our results provide for the first time, afunctional link between SLC1A5 activity and the growthdependency on glutamine observed in lung cancer cells. Thecytostatic consequences of targeting SLC1A5 activity couldbe explained in part by the inactivation of mTOR signalingin addition to the depletion of the intracellular glutamine

pool necessary for biosynthesis of macromolecules in lungcancer cells. The differential expression of SLC1A5, its cellsurface location, and its function as an amino acid trans-porter make it an attractive target in lung cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Hassanein, J.E. Clark, W.E. Alborn, P.P.MassionDevelopment ofmethodology:M.Hassanein,M. Shiota,W.E. Alborn, P.P.MassionAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Hassanein, M.D. Hoeksema, J. QianAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M. Hassanein, H. Chen, J.E. Clark, P.P.MassionWriting, review, and/or revision of the manuscript: M. Hassanein, J.E.Clark, W.E. Alborn, R. Eisenberg, P.P. MassionAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.D. Hoeksema, M. Shiota, B.K.HarrisStudy supervision: M. Hassanein, P.P. Massion

AcknowledgmentsThe authors thank SnjezanaZaja-Milatovic for her helpwith handling and

processing tissue and cell line arrays, Drs. Adriana Gonzalez for her help inthe pathologic classification of the tumors in our TMAs, and Brian Lehmmanfor his help with the siRNA experiments.

Grant SupportThis work was supported by Vanderbilt In vivo Cellular and Molecular

Imaging Center (ICMIC) Career Development Award (to M. Hassanein), aNCI grant CA102353 (to P.P. Massion), and a project in the VanderbiltSPORE in lung cancer (P50CA090949 to P.P. Massion).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 13, 2012; revised September 25, 2012; accepted November11, 2012; published OnlineFirst December 4, 2012.

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